A novel cell-based therapy for patients with aplastic anemia

Cytotherapy, 2010; 12: 678–683

A novel cell-based therapy for patients with aplastic anemia JIAYU CHEN1, WEIWEI LIU1, WEI YU1, LINGZHEN CHEN1, JINMING WU1, YU ZHAN1, LUPO WU2 & DEMAO YANG2 1Department

of Hematology, The 12th Municipal Hospital of Guangzhou, Guangzhou, China, and 2Zhongxing Yangfan Biotech Co. Ltd, Shenzhen, China

Abstract Background aims. Aplastic anemia (AA) is a rare but potentially life-threatening disease. There is a need for the development of new, more effective and less toxic therapies for treating AA. The safety and efficacy of an immune cell-based therapy for AA was examined. Methods. Thirty-one patients with idiopathic AA received intravenous infusions of ex vivo-activated autologous and allogeneic immune cells at least once a week. Response to therapy was assessed by symptoms, transfusion dependency, blood counts, bone marrow biopsy and survival. Results. Of the 31 patients, 25 (81%) had either complete (11, 35%) or partial (14, 45%) responses, while six (19%) showed no response to the therapy. The overall survival rates at 3 years were 90%. Conclusions. The therapy described appears to be safe and effective. The data from this pilot study suggest that a larger, controlled study is warranted. Key Words: aplastic anemia, cell therapy, hematopoiesis

Introduction Acquired aplastic anemia (AA) in humans is a rare but potentially life-threatening disease characterized by pancytopenia of the peripheral blood and aplasia of the bone marrow (BM) (1). The disease is either idiopathic or caused by exposure to toxic chemicals such as benzene (2). AA can be treated effectively with either allogeneic hematopoietic stem cell transplantation (HSCT) (3,4) or immunosuppressive therapy (5,6) with similar long-term survival rates. However, the majority of patients with AA in China are not eligible for HSCT because of a lack of HLA-identical siblings. Female patients before or at reproductive age are often concerned about irreversible gonadal damage induced by HSCT preparative chemotherapy. The immunosuppressive therapy, typically with horse anti-thymocyte and cyclosporine, restores hematopoiesis in approximately two-thirds of patients; however, disease relapse or secondary clonal disease is common (7,8). A recent retrospective analysis on 316 AA patients showed that only 41% and 53% patients with low absolute reticulocyte counts and absolute lymphocyte counts, respectively, responded to conventional immunosuppressive therapy and survived more than 5 years (9).

AA is more common in Asia than Europe and North America (10,11). Studies of immunosuppressive therapy and the large registry of BM transplantation data have revealed several differences in clinical outcome, including survival, response and quality of life among European patients (12,13). A study conducted in Japan showed that Japanese AA patients appeared to have lower response rates to primary and secondary immunosuppressive therapy than patients from Europe and North America (10), which may reflect effects of genetic background on the pathology of disease and/or response to treatment. We have developed a therapy based on ex vivo-activated peripheral blood mononuclear cells (PBMC) and successfully treated 14 patients with benzene-induced AA (14). In this study, all 14 patients had partial responses to the cell therapy at the time of publication and were found to be fully recovered later. In a mouse model under severe myelosuppression induced by chemotherapy and irradiation, the therapy was effective in enhancing survival and restoring normal hematopoiesis (15). Autologous and allogeneic PBMC have been found to be equally effective in previous animal and human studies (14,15). We performed an open-labeled, pilot

Correspondence: Demao Yang, PhD, Zhongxing Yangfan Biotech Co. Ltd, 1-211 Bio-Incubator, GaoXin C, 1st Avenue, Shenzhen Hi-Tech Industrial Park, Shenzhen, China 518057. E-mail: [email protected] (Received 23 April 2009; accepted 30 January 2010) ISSN 1465-3249 print/ISSN 1477-2566 online © 2010 Informa Healthcare DOI: 10.3109/14653241003695000

Cell-based therapy for AA trial of the therapy for treating idiopathic AA, to assess its safety and efficacy. Methods Patients The study was conducted at The 12th Municipal Hospital of Guangzhou (Guangzhou, China). The institutional review board of the hospital approved the study, and all patients provided written informed consent. Since 2001, 31 patients with idiopathic AA have been recruited and treated. The average age was 25 years (range 6–48 years) and the male/female ratio was 1.2:1. The time from diagnosis to treatment ranged from 1 to 10 years (mean 2 years). Of these patients, 17 patients (55%) had previously received but failed immunosuppressive therapies. We excluded patients with a current diagnosis or history of myelodysplastic syndrome, abnormal cytogenetics, Fanconi anemia and paroxysmal nocturnal hemoglobinuria. Patients were classified as having severe AA using a modification of the criteria of Camitta et al. (16). Severe AA was defined as two of three of the following peripheral blood criteria being present at the same time: neutrophils ⬍0.5 ⫻ l09/L, platelets ⬍10 ⫻ l09/L and reticulocytes ⬍10 ⫻ 109/L, in association with a hypocellular BM of which ⬍20% of cells were hematopoietic as assessed by BM biopsy. By these criteria, 19 (61%) had severe AA prior to therapy. Cells and cell culture Both allogeneic and autologous PBMC were used for the preparation of therapeutic cells. Allogeneic PBMC without an HLA match were obtained by leukopheresis from healthy donors, while autologous cells were from 20–50 mL venous blood, depending on a patient’s age. An HLA match for allogeneic cells was not required in the trial and thus no HLA typing was performed for donors. The PBMC were cultured under sterile conditions at approximately 4 ⫻ 106/mL for 48 h, in the presence of interleukin (IL)-2 (Ruixing Biopharmaceutical Inc., Beijing, China) at 500 IU/mL, granulocyte (G)–macrophage (M) colony-stimulating factor (CSF) (NCPC, Shijiazhuang, China) at 200 U/mL and calcium ionophore A23187 (Sigma, St Louis, MO, USA) at 100 ng/mL. Adherent cells were harvested by careful scraping using a rubber policeman; cell death as a result of scraping was less than 5%. The cells were washed three times with saline before infused intravenously to patients.

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Interventions At the beginning of the trial, five patients with severe AA received 6 ⫻ 105–1 ⫻ 108 ex vivo-activated allogeneic immune cells depending on a patient’s age per infusion per day for 4 consecutive days, followed by three infusions of autologous cells once a week. This cycle of infusions was repeated until a patient’s neutrophil count was greater than 0.5 ⫻ 109/L, followed by infusions of autologous cells once a week. Because of an unexpected shortage of allogeneic blood supply, the treatment scheme was changed to use allogeneic and autologous cells alternately and infusion was performed once a week. The use of allogeneic cells was primarily because of significant depletions of PBMC in patients with severe AA rendering sufficient harvesting of cells for the therapy difficult. All patients not classified as having severe AA received autologous cell therapy. Patients did not receive any immunosuppressive therapy during the trial. Results Clinical outcome During the past 7 years, 31 patients with AA have each received 27–108 therapies (mean 75), with a mean follow-up period of 3 years (range 1–7 years). Twenty-seven patients (90%) were alive after the course of therapy, while three patients (10%) had died of infection (patient 2) and cerebral hemorrhage (patients 1 and 3), respectively. The characteristics of the patients before the trial and their responses to the cell therapy are summarized in Table I. Physical strength, appetite and body weight were increased, while bleeding and infection episodes at an early stage of recovery were reduced in all responders. All responders showed significant improvement in hematopoietic activities in BM after the cell therapy, as assessed by BM biopsy examination; significant improvement was defined as increases in both cell density and numbers of proliferating hematopoietic cells in the BM were at least 3-fold. Patient 14 had received but failed immunosuppressive therapy with cyclosporine A before entering the trial. She developed significant hypertension and seizures requiring treatment in response to cyclosporine A. She responded well to the therapy and, despite not being fully recovered, gave birth to a naturally conceived and healthy baby 2 years after initiation of the cell therapy. Hematologic responses

Cell phenotype analysis Cell phenotype analyzes were performed by flow cytometry using standard protocols.

For severe AA, patients were classified as responders if they met one of the following two criteria: an absolute neutrophil count greater than 0.5 ⫻ 109/L

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Table I. Patient characteristics and responses to therapy. Before therapy

Patient number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

After therapy

Sex/age

ATG\CsA therapy

Blood transfusion

BM hypocellularity

Blood transfusion

Improvement in BM hematopoiesis

Response to therapy

M/28 F/24 M/31 M/9 M/22 M/48 F/21 M/27 M/8 F/15 F/41 M/30 M/32 F/26 M/31 F/42 F/12 M/32 M/19 M/28 F/25 F/6 F/27 F/33 M/25 F/25 M/20 M/22 M/28 F/24 F/22

ATG/CsA CsA CsA None None CsA CsA CsA CsA ATG/CsA CsA CsA CsA CsA CsA None ATG/CsA CsA None CsA None ATG/CsA None None None None None None None None None

Heavy Heavy Heavy Heavy Heavy Heavy Heavy Modest Modest Heavy Modest Heavy Heavy Heavy Heavy Heavy Heavy Heavy Heavy Heavy Modest Heavy Heavy Heavy Heavy Heavy Heavy Heavy Heavy Heavy Heavy

Severe Severe Severe Severe Severe Severe Severe Modest Severe Severe Modest Severe Severe Severe Modest Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe Severe

Heavy Heavy Heavy Heavy Heavy Heavy Heavy No No No No No No No No No No No No No No No No No No No No No No No No

No Modest No No No No Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant

NR NR NR NR NR NR PR PR PR PR PR PR PR PR PR PR PR PR PR PR CR CR CR CR CR CR CR CR CR CR CR

Numbers of infusions 27 42 32 105 60 22 104 55 90 108 72 70 100 65 100 84 60 88 80 75 85 70 65 95 72 75 90 65 105 75 80

M, male; F, female; ATG, anti-thymocyte globulin; CsA, cyclosporine A; PR, partial response; CR, complete response. Heavy blood transfusion indicates that patients received blood transfusions of either whole blood or packed red blood cells or platelets at a frequency of at least once every 10 days. Modest blood transfusion indicates that patients received blood transfusions at a frequency of no more than once a month. Severe BM hypocellularity indicates that cellularity in a patient’s BM was less than 20%, while modest hypocellularity indicates that cellularity was between 40% and 80%.

or a platelet count greater than 20 ⫻ 109/L. Partial remission was defined by transfusion independence or an unsupported increase of the counts of at least one cell line over baseline values (hemoglobin by at least 3g/dL, granulocytes by at least 0.5 ⫻ 109/L if previously lower than 0.5 ⫻ 109/L, platelets by at least 20 ⫻ 109/L if previously less than 20 ⫻ 109/L) and by doubling or normalization of counts of at least one cell line if previous counts of the respective cell line(s) did not meet the criteria for severe AA. All responses had to be confirmed by at least four blood counts at least 4 weeks apart. A complete response was defined as a normal blood count for age and sex together with independence from blood transfusion and normal BM histology. Of the 31 patients, six (19%) had no response, 14 (45%) had partial responses and 11 (35%) had complete responses to the cell therapy. Of 19

patients with severe AA, two (11%) died and two (11%) had no response, while five (26%) had complete responses and 10 (52%) partial responses. Ten of 12 patients who did not meet the definition of severe AA comprised six complete and four partial responses, while one patient (patient 2) died of septicemia and one patient had no response. Figure 1 shows the blood counts of white blood cells, absolute neutrophils, hemoglobin levels and platelets in all 31 patients before the cell therapy and at the last follow-up. Of 31 patients, 24 patients dependant on blood transfusion before the therapy became transfusion independent after the therapy. Of 25 responders, 13 patients who had varying degrees of reversed CD4/CD8 ratios (range 0.5–0.9) before the therapy showed restoration of normal CD4/CD8 ratios after the therapy.

Cell-based therapy for AA

The time needed to show significant improvement of peripheral blood counts varied considerably depending on the disease severity and individual patient. It often took 6–12 months for patients with severe AA, and 3–6 months for patients with less severe AA, to begin to show any improvement in white and red blood cell counts. Recovery in platelets was the most difficult and generally delayed for 6–12 months after restoration of white and red blood cells.

White Blood Cell Counts (109/L)

8

6

4

2

0 Before

After

Hemaglobin Levels (g/dL)

175

125

75

25 Before

After

250

Pleatlet Counts(109/L)

200

Side-effects Overall, the side-effects were temporary and well tolerated, with the most common being chills and fever between 37°C and 40°C, headache, nausea, vomiting and loss of appetite, usually infusion-related and temporary, and resolving within 6 h of cell infusion. Patients developing these symptoms were treated with conventional therapies accordingly. All patients who had complete responses remained disease free and all patients with partial responses continued to improve. To date, no disease relapse, graft-versus-host disease, myelodysplastic syndromes, paroxysmal nocturnal hemoglobinuria, leukemia or any other major secondary diseases associated with the therapy have been observed in the surviving patients.

150

Phenotype of infused cells

100

50

0 Before

After

5 Absolute Neutrophile Counts (109/L)

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4

3

2

1

0 Before

After

Figure 1. Blood counts of all 31 patients before and after the therapy.

The phenotypes of PBMC from five healthy donors before and after culture were analyzed by flow cytometry, and changes in expression of several major surface markers are summarized in Table II. The therapeutic cells consisted of a mixture of activated cells, including T cells, B cells, natural killer (NK) cells and CD14-positive and -negative monocytes. The phenotypes of autologous cells were difficult to generalize because of varying degrees of depletion of blood cells in patients; however, fluorescence-activated cell sorting (FACS) analysis was performed in three patients at mid (patient 1) and early (patients 2 and 3) stages of recovery and the results are shown in Table III. The major difference in phenotypic changes because of cell culture between normal donors and patients was that normal donors showed significant percentage increases in T-cell populations, while patients showed no such increases but rather slight decreases.

Time to response The restoration of normal blood count was a slow process and no patient with severe AA had a complete response in less than 1 year. Complete responses typically occurred 2–3 years after the initiation of the therapy.

Discussion In our pilot trial, we have demonstrated the safety and efficacy of a cell-based therapy for the treatment of some AA patients. The response and survival rates in AA patients who have received the therapy are

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Table II. Phenotypic analysis of therapeutic cells before and after cell culture. Surface markers CD3 CD3 ⫹ CD4 CD3 ⫹ CD8 CD19 CD16 ⫹ CD56 CD3 ⫹ CD4/CD3 ⫹ CD8 CD14 ⫹ CD45

Cell type Total T cells T-helper cells Cytotoxic T cells B cells NK cells Helper/cytotoxic T-cell ratio Monocytes

Before (%) 57 34 22 18 8 1.55 20

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

5 3 2 3 2 0.04 3

After (%) 66 41 27 8 3 1.50 11

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

6 4 3 3 1 0.03 5

Changes ↑ ↑ ↑ ↓ ↓ ↓ ↓

PBMC were taken from five healthy donors and surface lineage markers of four major subsets of blood cells, including T-helper and T-cytotoxic cells, B cells, NK cells and monocytes, were analyzed by FACS using a standard procedure. All numbers are mean ⫾ SEM.

comparable with those of conventional immunosuppressive therapies. The mechanism of the therapy for AA is not completely clear. Our hypothesis is that ex vivo-activated immune cells produce multiple known and unknown potent hematopoietic cytokines; working in concert, these cytokines help growth and differentiation of blood stem cells. Previous study has shown that therapeutic cells produce and secrete many cytokines, including some potent hematopoietic factors such as G-CSF, IL-6 and IL-8 (15). Moreover, most therapeutic cells home to liver, spleen and BM, where cytokines are released on target cells at close range. We are now carrying out a series of experiments to study the mechanism of the therapy that will hopefully lead to a better understanding of the pathology of AA. AA is considered to be an autoimmune disease, in which autoimmune T cells kill blood stem cells and other progenitor cells. We have tried but failed to find evidence that the therapy may be involved

in correcting the autoimmunity. CD4 and CD25 surface markers for regulatory T cells, IL-2 and interferon (IFN)-γ secreted by T cells from patients were monitored in some responders before, during and after the therapy; however, no significant differences were found. Previous findings (14,15) have shown that both allogeneic and autologous immune cells are effective in treating benzene-induced AA patients, and mice with severe myelosuppression induced by chemotherapy and irradiation, suggesting that the effects of the therapy are not through an HLA-mediated mechanism. In a later mouse model, exnogeneic human cells were also found to be effective (15), further supporting our hypothesis that cytokines rather than precisely regulated immune responses are key for the therapeutic effects. There was no general suppression of the immune system, such as increased infection rates, as a result of the therapy in our patients, suggesting that the mechanism of this therapy could be very different from that of conventional immunosuppressive therapy.

Table III. Phenotypic analysis of therapeutic cells from three patients before and after cell culture. Surface markers

Cell type

CD3

Total T cells

CD3 ⫹ CD4

T-helper cells

CD3 ⫹ CD8

Cytotoxic T cells

CD19

CD16 ⫹ CD56 CD3 ⫹ CD4/CD3 ⫹ CD8

B cells

NK cells

Helper/cytotoxic T-cell ratio

Patient

Before (%)

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

58 69 59 33 25 27 22 31 28 20 14 6 19 13 31 1.5 0.82 0.89

After (%) 53 66 55 29 23 21 20 25 30 19 4 2 20 19 36 1.4 0.89 0.70

PBMC were taken from three AA patients under cell therapy and surface lineage markers of three major subsets of blood cells, including T-helper and T-cytotoxic cells, B cells and NK cells, were analyzed by FACS using a standard procedure. Patient 1 was partially recovered while the other two were in early stages of recovery.

Cell-based therapy for AA It is interesting that plant mitogens of phytohemagglutinin-L (PHA-L) and pokeweed migogen (PWM) have been reported to be successful in treating patients with aplastic anemia and cancer (17–19). The authors suggested that mitogens stimulate production of growth factors and thereby hasten recovery from aplasia, and that mitogens somehow suppress the autoimmunity causing AA. The approaches of plant mitogen use and our cell therapy share a striking similarity in the non-specific activation of immune cells. These factors should be considered seriously for studying the mechanism of cell therapy and pathology of AA. One of the shortcomings of the therapy was the slow recovery in the AA patients, and we are currently studying various methods to improve it. Although this study was small and uncontrolled, the data suggest that the therapy may provide a potential alternative for AA patients who do not respond to conventional therapies and have no suitable donors for allogeneic HSCT. Disclosure of interest: The authors report no potential conflicts of interest. References 1. Young NS. Acquired aplastic anemia. Ann Intern Med. 2002;136:534–46. 2. Young NS: Drugs and chemicals, in Young NS, Alter BP (eds): Aplastic Anemia, Acquired and Inherited, Philadelphia, W.B. Saunders, 1994, p 100–32. 3. Deeg HJ, Leisenring W, Storb R, Nims J, Flowers ME, Witherspoon RP, et al. Long-term outcome after marrow transplantation for severe aplastic anemia. Blood. 1998;91:3637–45. 4. Bacigalupo A, Brand R, Oneto R, Bruno B, Socié G, Passweg J, et al. Treatment of acquired severe aplastic anemia: bone marrow transplantation compared with immunosuppressive therapy. The European Group for Blood and Marrow Transplantation experience. Semin Hematol. 2000;37:69–80.

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